Regulation of the immune response to both self-antigens and foreign pathogens is integral to host survival. The natural regulatory pathways involved may also afford the opportunity to intercede in undesirable immune responses such as those occurring to allogeneic transplants. The attractiveness of this approach is evident in the enthusiasm for applying regulatory T cells (Tregs) to promote transplant survival in patients. Recent evidence suggests that parallel regulatory pathways may exist in the humoral arm of the immune response though the exact cells involved and the mechanisms of regulation are yet to be fully characterized. Despite the convincing evidence for regulatory B cells (Bregs) in non-transplant settings (autoimmunity, infectious disease, and bone marrow transplant/GVHD), a clear role for B cells or Bregs in the maintenance of induced transplant tolerance has not yet been well-established. During the last funding period we made the unexpected observation that a well-studied model of antibody induced transplantation tolerance depends on the presence of B lymphocytes and that graft prolongation in the model was transferrable to secondary hosts by TIM-1+ B cells, demonstrating Breg activity. In the proposed studies, we will characterize the contribution of Bregs to transplantation tolerance using models developed in the previous funding period. In Aim I, we focus on the mechanisms by which Bregs suppress the response of nave Teff and examine Breg antigen specificity, characterize Breg induced changes in the character of the immune response and define the role of immunomodulatory cytokines (IL-10 and TGF-). In Aim II, we will take advantage of our finding that anti-CD45RB-induced tolerance is dependent on both Tregs and B cells to explore the possibility that Treg development is fostered by Bregs or that Tregs and Bregs collaborate to promote graft acceptance. Finally, Aim III examines the contribution of the costimulatory molecule TIM-1 to tolerance induced by anti-CD45RB and will rely on a new TIM1-/- line generated by our collaborator, Dr Terry Strom. Collectively, these studies will further our understanding of the unique contribution of regulatory B cells to transplantation tolerance.